Leishmania Promastigotes Require Opsonic Complement to Bind tothe Human Leukocyte Integrin Mac-1(CD11b/CD18)DavidM . Mosser,* Timothy A. Springer, § and Michael S. Diamond11§*Department of Microbiology and Immunology, Temple University School ofMedicine, Philadelphia, Pennsylvania 19140 ; andII Committee on Cell and Developmental Biology, and *Department of Pathology, § The Center for Blood Research andHarvard Medical School, Boston, Massachusetts 02115

Abstract . Previous reports have suggested that Leish-mania spp . interact with macrophages by binding toMac-1 (CDllb/CDl8), a member of the leukocyte in-tegrin family. To better define this interaction, wetested the ability of leishmania promastigotes to bindto purified leukocyte integrins and to cloned integrinsexpressed in COS cells . We show that leishmania pro-mastigotes bind to cellular or purified Mac-1 but notlymphocyte function-associated antigen-1 in a specific,dose-dependent manner that requires the presence ofserum. Binding is inhibited with specific monoclonalantibodies to Mac-1. In the absence of complement

THE integrins comprise a family of structurally relatedcell surface receptors that coordinate a network ofcell-cell and cell-extracellular matrix interactions

(Springer, 1990; Hemler, 1990) . Lymphocyte function-associated antigen-1 (LFA-1; l CDl la/CD18), Mac-1 (com-plement receptor type 3 [CR3] ; CDllb/CD18), and p150,95(CDllc/CD18) constitute a subfamily of integrins known asthe leukocyte integrins (Kishimoto et al ., 1989) . These re-lated glycoproteins, which are critical for the adhesive eventsin the immune system, structurally share a common ß sub-unit that is noncovalently associated with unique a chains .LFA1, which is expressed on all leukocytes, mediates

lymphocyte adhesion to endothelial cells (Dustin andSpringer, 1989), facilitates the T cell conjugation that is re-quired for antigen-specific killer and helper functions (Da-vignon et al ., 1981), and promotes the adhesion required fornatural killing (Krensky et al ., 1983) . These adhesive inter-actions occur via at least two characterized counter recep-tors, ICAM-1 (Rothlein et al ., 1986 ; Marlin and Springer,1987) and ICAM-2 (Staunton et al ., 1989 ; de Fougerolleset al ., 1991) . p150,95 is restricted in distribution to myeloidcells and a small population of T and B lymphocytes . Itsfunction is not clear, but previous studies have suggested its

1 . Abbreviations used in this paper: C3, third component of complement;C8D, serum from an individual genetically deficient in the eighth compo-nent ofcomplement ; CR3, complement receptor type3 ; LFA-1, lymphocytefunction-associated antigen-1; LPG, lipophosphoglycan .

© The Rockefeller University Press, 0021-9525/92/01/511/10 $2 .00TheJournal of Cell Biology, Volume 116, Number2, January 1992511-520

opsonization, three different species of leishmaniatested fail to bind directly to any of the three leuko-cyte integrins . We show that binding to Mac-1 requiresthe third component of complement (C3) . Organismsincubated in heat-inactivated serum or serum that hasbeen immunologically depleted of C3 fail to bind toMac-1 . Because the addition of purified C3 to C3-depleted serum restores leishmania binding to Mac-1,we suggest that parasites gain entry into macrophagesby fixing complement and subverting a well-character-ized adhesive interaction in the immune system betweenMac-1 and iC3b .

involvement in the binding of iC3b-opsonized particles (My-ones et al ., 1988), in the adhesion of cells to endothelium(Keizer et al ., 1987a,b ; to Velde et al ., 1987), and in cellularadhesion to substrates coated with serum proteins (Ander-son et al ., 1986 ; Loike et al ., 1991) . Mac-1 is primarily ex-pressed on myeloid and natural killer cells, and is responsi-ble for myeloid cell adhesion to endothelial cells, neutrophilhomotypic aggregation, and macrophage phagocytosis offoreign particles that are opsonized by complement (Kishi-moto et al ., 1989) . Mac-1 achieves these functions by bind-ing to a multiplicity of cellular and soluble ligands, includingICAM-1 (Smith et al ., 1989 ; Diamond et al ., 1990), iC3b(Beller et al ., 1982 ; Wright et al ., 1983), fibrinogen (Altieriet al ., 1988, Wright et al ., 1988), and factor X (Altieri andEdgington, 1988) .

Recently, several groups have reported that leukocyte ad-hesion receptors on macrophages can mediate the directrecognition ofmicrobes in the absence ofexogenous comple-ment. Monoclonal antibodies to one or a combination oftheleukocyte integrins have been shown to inhibit the phagocy-tosis of a diverse array of unopsonized microbial pathogens .These include Leishmania major (Mosser and Edelson,1985 ; DaSilva et al ., 1989 ; Cooper et al ., 1988), Leish-mania donovani (Blackwell et al ., 1985), Leishmania mexi-cana (Talamus-Rohana et al ., 1990), Bordatella pertussis(Relman et al ., 1990), Histoplasma capsulatum (Bullockand Wright, 1987), Saccharomyces cermisia (Ross et al .,1985 ; Tàmato et al ., 1989), Escherichia coli (Wright and

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Jong, 1986), and Mycobacteria leprue (Schlesinger and Hor-witz, 1990) . In some of these studies, macrophages bounddirectly to these organisms in vitro in the absence of exog-enous complement, and this binding was inhibited with anti-bodies to the CD18 family ofadhesion receptors . The mech-anism(s) for this direct recognition remains unclear. Mac-1has been implicated in the binding of saccharides (Ross etal ., 1985) and polysaccharides (Wright and Jong, 1986)which may be present on the surface of various microbes .Mac-1 is also reported to bind directly to two leishmania sur-face structures, the lipophosphoglycans (Tàlamus-Rohana etal ., 1990) and the major surface protease gp63 (Russell andWright, 1988) . In contrast, others (Ezekowitz et al ., 1985 ;Blackwell et al ., 1985) have argued that the recognition ofmicrobes by Mac-1 depends on C3, which may be derivedfrom the macrophage itself. To address whether the leuko-cyte integrins bind directly to endogenous parasite surfacestructures, or indirectly via complement components, wehave investigated the binding of leishmania to transfectedfibroblasts expressing leukocyte integrins and to purified leu-kocyte integrin substrates . In this work we show that the hu-man leukocyte integrins do not support leishmania bindingdirectly, but rather require opsonic complement . Further-more, we show thatwhen complementis present onL . major,thebinding ofthese organisms is mediated almost exclusivelyby the Mac-1 integrin .

Materials and Methods

LeishmaniaThe U.S. National Institutes of Health S strain of L. major (Bjorvatn andNeva, 1979) was isolated from a West African patient with cutaneous leish-maniasis and was originally provided by Dr. D. Wyler, Tufts UniversityMedical Center (Boston, MA) . The Josefa strain ofL. mexicana amazonen-sis (Mosser et al ., 1986) was isolated from a Brazilian patient with persis-tent cutaneous leishmaniasis and was provided by Dr. Janet Keithly, NewYork State Public Health Center (Albany, NY) . The 1S strain ofL. donovani(Murray, 1981) was isolated from a Sudanese patient with kala azar and wasprovided by Dr. Henry Murray, Cornell Medical School, New York. Pro-mastigotes were cultured in Schneider's complete drosophila mediumto sta-tionary phaseas described (Mosser et al ., 1986) and radiolabeled with triti-ated uracil overnight as described (Mosser and Edelson, 1985) .

MonoclonalAntibodiesThe following murine monoclonal antibodies (mAbs) were used dilutedfrom ascites : LPM19c (IgG2a, anti-CDllb, a gift of Dr. K . Pohlford, Ox-ford, England) (Uciechowski and Schmidt, 1989), 1413611.2 (IgGI, anti-CD1 lb, a gift ofDr. L. K. Ashman, Adelaide, Australia) (Uciechowski andSchmidt, 1989), 904 (IgGI, anti-CDllb, a gift of Dr . J. Griffin, Boston,MA) (Letvin et al., 1983), and TSl/22 (IgGI, anti-CD1la) (Sanchez-Madridet al., 1982) . The following mAbs were used as purified IgG : R6.5 (IgG2a,anti-CD54, a gift of Dr. R . Rothlein, Boehringer-Ingelheim, Ridgefield,CT), YZ.1 (IgGI, anti-CRI, a gift ofDr. R . Jack, Boston, MA) (Changelianet al ., 1985), LM2/1 (IgGI, anti-CDllb) (Miller et al ., 1986), 60.3 (IgG2a,anti-CD18) (Beatty et al ., 1983), OKMI (IgG2a, anti-CDI lb) (Wright et al .,1983), IB4 (IgG2a, anti-CD18) (Wright et al ., 1983), and SHCL3 (IgG2b,anti-CDllc) (Schwarting et al ., 1985) .

Monocyte-derived Human MacrophagesMonocytes were isolated from peripheral blood using "Lymphoprep"(Nycomed Pharma, Oslo, Norway) according to the manufacturer's instruc-tions. They were incubated in RPMI 1640 supplemented with penicillinG/streptomycin (100 U/ml and 100 pg/ml, respectively), 2 mM glutamine,and 5% autologous human serum, in Teflon beakers for 4 d as described(Speert et al ., 1988) . Cells were adhered to 13-mmroundcoverglasses over-night in the presence of 5 % autologous serum, washed the following dayin serum-free phagocytosis buffer, andused in the leishmania-binding assay.

The Journal of Cell Biology, Volume 116, 1992

Integrin-coated SubstratesHuman leukocyte integrins were purified as previously described (Diamondet al ., 1990 ; Dustin and Springer, 1989 ; Slacker and Springer, 1991) .Briefly, peripheral blood leukocyte lysates or hairy cell leukemia spleno-cytes were solubilized in Triton X-100 and the integrins were purified byimmunoaffinity chromatography. Peak fractions ofimmunoaffinity-purifiedLFA-1, Mac-1, and p150, 95 protein ata starting concentration of 3-5 pg/mlin 1% octyl glucoside were diluted 1 :12 in 50 mM Tris-HCI, pH 8.0, 150mM NaCl, 2 mM MgCl, and added to nontissue culture-treated 96-wellplates (Flow Laboratories, Inc ., McLean, VA) for 90 min at room tempera-ture . Plates were subsequently blocked in PBS, 2 mM MgCIZ, 1% BSA for1 h at 37°C and stored at 4°C until further use in the presence of 0 .025 %NaN3 and 50 14g/ml gentamicin .

Leishmania-binding AssayRadiolabeled leishmania promastigotes were resuspended in phagocytosisbuffer which consists of equal parts of DME and TC199 buffered with 25mM Hepes supplemented with 1% BSA . Parasites were added to eitherpurified integrins on 96-well plates or to macrophages or transfected COScells on 24-well plates for60 min at 37°C. For assays performed in the pres-ence of serum, a final concentration of 5 % serum from a patient with adeficiency in the eighth componentofcomplement, C8D (Mosser and Edel-son, 1987), was added to 5 x 107 parasites for 15 min before their additionto cells or to Mac-l-coated substrates. C8D was used to avoid experimentalartifacts due to complement-mediated lysis, whichcauses the release of freeuracil from radiolabeled parasites (Mosser and Edelson, 1984b) . After a1-h incubation with radiolabeled parasites, plates were washed extensivelyusing a25-gauge needle aspiration aparatus with warm (35°C) phagocytosisbuffer. Bound parasites were solubilized in 0.5% Triton X-100 and 0.1 MNaOH . After neutralization with 0.1 M HCI, lysates were analyzed ina tri-carb scintillation counter (Packard Instrument Co. Inc., DownersGrove, IL) .

To inhibit parasite binding to Mac-1 plates, purified lipophosphoglycan(LPG) from L. major was added to Mac-1 plates at a final concentrationof 100 jg/ml for 15 min before and during the addition of leishmania . Toinhibit leishmania C3 opsonization, increasing concentrations ofLPG wereadded to a total of 1 x 101 promastigotes in 5 % C8D for 15 min . LPG(McConville et al ., 1987) was a gift of Emanuela Handman (Melbourne,Australia) .

COS Cell TlransfectionsCOScells were transfected in 10-cm tissue culture-treated plates with CDl land CD18 cDNA as previously described (Diamond et al ., 1990) . Briefly,cells at -50-60% confluency were transfected with 6 /+g each of CDl l andCD18 cDNA by the DEAF-Dextran method for 4 h at 37°C (Kingston,1987) . 72 h after transfection, cells were removed from tissue culture plateswith PBS containing 5 mM EDTA and plated onto 13-mm round coverslipsin 24-well plates overnight . Cells were assayed the following day, 4 d aftertransfection. Flow cytometry showed that -40-60% of LFA-1-transfectedcells were positive for LFA-1 expression, and that 30-40% of Mac-l-trans-fected cells were positive for the expression of Mac-1 .

Flow CytometryIndirect immunofluorescence and flow cytometry were performed as previ-ously described (Diamond et al ., 1990) .

SDS-PAGEProtein samples were analyzed on reducing (5 % ß-mercaptoethanol)SDS-5% polyacrylamide gels (Laemmli, 1970) and silver stained as de-scribed (Diamond et al ., 1990) .

Results

Leishmania Promastigotes Bind to HumanMonocyte-derived MacrophagesIncreasing concentrations of radiolabeled L. major pro-mastigotes were added to human monocyte-derived macro-phages taken from three donors in either the presence or ab-

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sence of 5% fresh human C8-deficient serum (C8D) . Aparasite dose-response curve (Fig . 1 A) shows that promasti-gotes bind to these cells more efficiently in the presence ofserum than in its absence . At the highest parasite inputconcentration (2 .5 x 10 6 ), which in this experiment corre-sponds to a parasite/macrophage ratio of50:1, macrophagesin the presence of serum bound an average of between twoand three parasites per cell . In the absence of serum at thisdose, these macrophages bound <0.5 parasites per cell . Thethree donors depicted in Fig . 1 were representative of a totalof six donors examined . Five of the six donors showed acomparable lack of binding in the absence of serum at alldoses tested, and at least a fivefold increase in binding in thepresence of serum. A single donor showed greater (approxi-mately twofold) serum-independent binding relative to theother donors tested in parallel . The low binding of L. majorto human macrophages in the absence of exogenous comple-ment was not improved by pretreating the monolayer withagents that activate Mac-1, including either 0.2 Ag/ml PMA(data not shown) or 10 pM ATP (Altieri and Edgington,1988) (Fig . 1 A) . The inefficient binding in the absence ofserum confirms and extends a previous observation (DaSilvaet al ., 1989), and establishes that the parasites used in thisstudy were in the infective stage.Most of the serum-dependent binding of promastigotes to

human macrophages was inhibited by mAbs to Mac-1, butnot by antibodies to either of the other two leukocyte inte-grins (Fig . 1 B) . OKM1, a Mac-1 a chain-specific mAb,inhibited binding by -50% . The ß chain-specific antibody60.3 inhibited binding by 75 %, and a combination of eithertwo Mac-1 a chain antibodies (LPM19C and OKMI) or ana and a ß (OKMI and 60.3) inhibited binding by >80% .Antibodies to either LFA-1 (TSI/22) or p150,95 (SHCL3) in-hibited binding by <15% . An antibody to CRI, YZ.1, alsohad little inhibitory effect in this system (data not shown) .

Mosser et al . Leishmania Require Complement to Bind to Mac-] 51 3

PROMASTIGOTES ADDED (x10-6)

PROMASnGOTE BINDING (% Control)

Figure 1. The binding ofradiolabeledL. majorpromastigotes to human monocyte-derivedmacrophages . (A) Dose-response curve ofL. ma-jor binding to human macrophages. Increasing concentrations of leishmania promastigotes were added to 24-well plates containing N5 x10° macrophages per well . In this representative experiment (of six donors, each analyzed twice), cells from three different donors (o,0, and t,) were incubated with promastigotes in either the presence (closed symbols) or absence (open symbols) of 5% fresh autologousserum . Standard deviations are shown if they are larger than symbols . Macrophages from one individual (T) were pretreated for 15 minwith 10 /1M ATP before the addition of2 .5 x 106 promastigotes under serum-free conditions . (B) The inhibition ofpromastigote bindingto human macrophages by mAbs to Mac-1 . Monocyte-derived macrophages were preincubated with either a 1 :100 dilution of ascites(LPM19C, 904, TSl/22) or 10 fcg/ml purified antibody (60.3, SHCL3, OKMI, 134) for 20 min at 35°C . A total of 2 x 106 radiolabeledpromastigotes were then added for 60 min at 35°C . Counts per minute were determined in triplicate and compared to simultaneous controlcounts per minute (no antibody) . Results reported here are the mean percent of at least two experiments done in triplicate.

Leishmania Promastigotes Adhere Only toImmobilizedMac-1Purified leukocyte integrins were isolated by immunoaffinitychromatography and immobilized on plastic . The three inte-grins studied contain a common M 95,000 ß chain whichnoncovalently associates with three a subunits of 177,000,165,000, and 150,000 M, (CDlla-c) (Fig . 2) . Slight differ-

Figure 2. Silver-stained SDS-PAGE of affinity-purified leu-kocyte integrins . PurifiedLFA1(15 11) from a TS2/4 mAb af-finity column, purified Mac-1(15 jl) from an LM2/1 mAbaffinity column, and purifiedp150,95 (10 j1) from a CBR-p150/4G1 mAb affinity column(lanes 1, 2, and 3, respec-tively), were subjected to re-duction, SDS-5 % PAGE andsilver staining . Molecularweight standards are markedto left .

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Figure 3. The binding of leishmania promastigotes in 5% serum to plates coated with purified human leukocyte integrins . A total of 2x 106 radiolabeled (A) L. major or (B) L. amazonensis promastigotes were added to 96-well plates coated with purified Mac-1, p150,95or LFA-1 as described in Materials and Methods. After washing the average cpm/well f SD of triplicate determinations (left axis) andthe deduced average number oforganisms/well (right axis) were determined . The data shown here for L. major are representative of threeseparate experiments, and that for L. amazonensis are representative of two experiments .

ences in the relative migration of the R chains shown in thissilver-stained SDS-polyacrylamide gel occur because differ-ential subunit association affects the site-specific glycosyla-tion of the common R chain (Dahms and Hart, 1986) . Theseisolated receptors retain their immunologic epitopes andfunctional activity. The receptors when adsorbed to plasticretain an active conformation as they bind their respectiveligands, including iC3b, ICAM-1, and ICAM-2 (Dustin andSpringer, 1989 ; Diamond et al ., 1989 ; de Fougerolles et al .,1991), without any addition of exogenous stimuli . They alsobind mAbs against activation-specific neoepitopes (Diamond,M. S., and T . A . Springer, unpublished observations) .Binding of promastigotes in 5 % serum occurred to a

significant level with Mac-l-coated substrates (Figs . 3 and4) . The parasites did not bind efficiently to either p150,95 orLFA-1, even though integrin site densities used were able tosustain stimulated endothelial cell adhesion when tested inparallel (data not shown) . The binding of parasites topurified Mac-1 was strong, as 20% of the input ofL. majoradhered to Mac-l, whereas 2-5 % bound to p150,95 and 1

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attached to LEA-1 or the control BSA. At the maximum para-site input (2 x 106 parasites/well) the total number of or-ganisms bound to Mac-1 plates in four independent assayswas 4.6 x 105 t 3.5 x 10° organisms per well, forming anear-confluent lawn of parasites which adhere to the Mac-1substrate despite the high shear forces created by the washingprocedure (Fig . 4) . This pattern ofMac-l-dependent bindingwas reproduced in a second leishmania species, L. amazo-nensis (Fig . 3 B) .

In the presence of serum, leishmania bind to plates coatedwith Mac-1 in a parasite dose-dependent manner (Fig. 5 A) .In this experiment between 20 and 25% of the total orga-nisms added to the plates at each dose remained adherent tothe wells after washing . Under serum-dependent conditionsin four independent assays, the percentage of the parasite in-put which adhered to Mac-1 plates ranged from 12 to 26%,with a mean of 20% . In the absence of serum, the numberofparasites bound to the Mac-l-coated substrate was similarto that bound to BSAcoated plates .

Parasite binding to Mac-1 plates was also dependent on the

Figure 4. Photomicrograph of L. major promastigotes bound to Mac-l-coated plates . A total of 2.5 x 106 radiolabeled leishmania wereadded to purified Mac-1- or LFA1-coated plates in the presence of 5% serum for 60 min at 35°C . This inverted micrograph shows viablepromastigotes adhered tightly to a Mac-1 plate (A) but not to LFA-1 (B) after extensive washing .

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A.

PROMASTIGOTES ADDED (x10-5)

MAC-1 CONCENTRATION (reciprocal dilution)

Figure 5. The binding of L. major promastigotes to purified Mac-l-coated plates. (A) Increasing concentrations of radiolabeled leishmaniain the presence (o) or absence (o) of5 % C8D were added for 1 h at 35°C to 96-well plates coated with Mac-1 . These data are representa-tive oftwo experiments . Data points represent the average of triplicate determinations f SD which are shown only when larger than thesymbols . In this experiment leishmania incorporated 0.012 cpm/organism, thus the maximal binding of 5,771 cpm equals a total of 4 .8x 105 1eishmania bound per well . Background binding of leishmania to parallel BSA-coated wells, which was 91 cpm at the highest para-site input dose (o), was not subtracted from any of the values . (B) The binding ofleishmania promastigotes to plates coated with decreas-ing amounts of human Mac-1 . A constant amount of radiolabeled L . major promastigotes (2 x 106) was added to plates coated with in-creasing dilutions of purified Mac-1 antigen . Mac-1 was diluted from 1 :12 to 1:30 in 50 mM Tris, 150 mM NaCl, 2 mM MgCl, whichcorresponds to a ten-fold range of Mac-1 site densities . A 1 :12 dilution of Mac-1 was previously determined to give maximal parasite bind-ing. The binding of leishmania to control wells coated with BSA is included (T) .

quantity ofthe Mac-1 substrate . A constant amount (2 x 106)of leishmania were added to plates, with increasing dilutionsof Mac-1 immobilized to plastic wells (Fig . 5 B) . Over arange of Mac-1 dilutions, parasite binding decreased from amaximum of2.3 x 105 parasites/well to levels indistinguish-able from background (Fig . 5 B) .

Adhesion ofLeishmania to Mac4 Plates RequiresOpsonic Complement

Mosser et al . Leishmania Require Complement to Bind to Mac-1

The binding of promastigotes to Mac-1 plates is dependenton serum complement . Threedifferent species ofleishmania-L. major, L. amazonensis, and L. donovani-were added toMac-1 plates either in the presence of C8D, in the presenceof heat-inactivated C8D (56°C, 60 min), or in the absenceof serum . All the species tested bound to Mac-1 plates onlywhen fresh serum containing opsonic complement was pres-ent (Fig . 6, A-C) . In the case of L. major, either heat inacti-vation of serum or immunologic depletion of C3 from serumdecreased binding from peak levels (10,259 cpm) to back-ground (289 and 364 cpm, respectively) . Furthermore, pre-treatment of serum with agents that are known to biochem-ically inactivate C3 or prevent C3 deposition inhibitedparasite binding to Mac-1 by >80% (Table I) . The sametrend was observed with the other two species tested as well .The binding of L. amazonensis to Mac-1 in the presence of5 % C3-depleted serum (OD) was 661 f 206 cpm. The ad-dition of 250 p.g/ml of purified 0 to this serum (Quidel,San Diego, CA) partially restored binding to 3,692 f 1114cpm (Fig. 6 B) . In a separate experiment, the addition ofpurified C3 to OD partially restored binding of L. majorfrom 472 f 76 cpm to 1,509 f 346 cpm .

The Binding ofPromastigotes to M"-1 Substrates IsSpecific and Inhibited by mAbsPromastigotes were added to Mac-1 plates which had been

B .

preincubated for 20 min with various mAbs (Fig. 7) . TwomAbs to Mac-1, LPM19C and 14B6E.2, each inhibited bind-ing by >80 %, while a third mAb, LM2/1, inhibited bindingby <30% . OKMI inhibited binding by 53 % . A combinationof OKMI and LPM19C inhibited binding to levels indistin-guishable from background . An isotype matched antibodyR6.5, against a control antigen, ICAM-1, failed to inhibitparasite binding. Interestingly, two n Abs (904 and IB4)which have been reported to inhibit LPG binding to Mac-1but not affect iC3b binding to Mac-1, had little, ifany, effectin our binding assay.

Leishmania LPGDoes Not Directly CompeteforMac-1 BindingBecause other groups report a role of leishmania LPG in thebinding of leishmania to macrophages (Thlamus-Rohana,1990 ; Handman and Goding, 1985), we studied its role inour defined system . LPG from L. major promastigotes (10014g/ml) was preincubated with Mac-l-coated plates for 15min before the addition of increasing concentrations ofpreopsonized leishmania . LPG did not inhibit parasite bind-ing . Leishmania bound to LPG-treated Mac-1 plates as wellas they bound to control plates (Fig . 8 A) . However, if leish-mania were incubated simultaneously with LPG and com-plement at the time of opsonization, LPG partially inhibitedthe binding of parasites to Mac-1 in a dose-dependent man-ner (Fig . 8 B) . This inhibitory effect was overcome by theaddition of excess (20%) serum . In this system, LPG in-hibited the binding of leishmania to Mac-1 only when addedtogether with complement, presumably by competing withparasites for complement deposition .

Promastigote Binding to COS Cells ExpressingCloned Leukocyte IntegrinsWhile the adhesion assays of leishmania promastigotes to

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purified leukocyte integrins strongly suggested an interac-tion with Mac-1, we wanted to confirm this in a cellular sys-tem . To achieve expression of only a single leukocyte inte-grin, COS cells were cotransfected with cDNAs for thecommon ß subunit and either the Mac-1 or LFA-1 « subunit .These cells were used in a leishmania promastigote-bindingassay 4 d after transfection . At this time surface expressionof LFA-1 ranged from 40 to 60%, while the expression ofMac-1 ranged from 30 to 40%, as determined by flow cytom-etry (data not shown; Diamond et al ., 1990) . A range ofleishmania doses was added to these cells in either the pres-ence or absence of exogenous complement .COS cells transfected with Mac-1 showed greater binding

of parasites than did mock-transfected cells (Fig . 9) . In con-trast, the binding ofL . major to LFA-1-transfected COS cellswas similar to that ofL. major to mock-transfected cells. Theincreased binding of leishmania to Mac-l-expressing COScells, as in the purified protein experiments, required thepresence of serum . This increase in parasite binding wasspecific to Mac-1, as an antibody to Mac-1 but not to LFA-1inhibited binding to levels obtained without serum . Micro-scopic observation (Fig . 10) reveals that between 30 and40% of the cells bound larger numbers of parasites (>10),whereas the remaining cells bound only 1-2 organisms . Thisfraction of the cells which sustain strong parasite bindingcorrelates well with the profile of Mac-1 expression seen byflow cytometry and suggests that cells expressing highamounts of Mac-1 bind significantly greater numbers ofparasites than do transfected cells which do not expresssignificant levels of Mac-1 .

51 6

Discussion

Figure 6. The binding of (A) L. major, (B) L. amazonensis, or (C) L .donovani promastigotes to purified Mac-l-coated plates . Organisms wereincubated in the presence of C8D, in heat-inactivated normal human se-rum (H.L ), in the absence of serum (S.L ), in C3D, or in C3D withpurified C3 added back to it (C3D+C3) . The mean cpm's bound per wellf SD of triplicate determinations (left axis) and the average total numberof organisms bound per well deduced from these cpm's (right axis) is in-cluded for each species .

Leishmania are obligate intracellular parasites that reside al-most exclusively within mononuclear phagocytes (Chang,1983) . Several groups have examined the molecular basis forthe binding and internalization of promastigotes by macro-phages in vitro and have demonstrated that specific macro-phage phagocytic receptors mediate this interaction (Mosserand Edelson, 1984b ; DaSilva et al ., 1989 ; Blackwell et al .,1985 ; Wilson and Pearson, 1988 ; Chang, 1983; Tàlamus-Rohana et al ., 1990) . Previous studies have shown that leish-mania are able to fix complement directly from nonimmune

Table L The Binding ofRadiolabeledLeishmania to Mac-] Plates after Incubationsin Serum Depleted of C3

" C8D was pretreated for 30 min at 35°C with 5 U/ml cobra venom factor, 0.2M potassium thiocyanate, 0.2 M hydroxylamine, or 15 mM EDTA.t Counts per minute of radiolabeled leishmania bound to Mac-1 plates afterwashing t standard deviation of triplicate samples.

A. L MAJOR 0. L AMAZONENSIS

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Untreated 2,402 t 268 -CoVF 471 t 8 80KSCN 236 t 106 90Hydroxylamine 491 t 289 80EDTA 216 t 13 91C3D 207 t 5 91No serum 219 t 18 91

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serum (Mosser and Edelson, 1984b ; Puentes et al ., 1988),and that this enhances entry into (Mosser and Edelson,1985) and survival within macrophages (Mosser and Edel-son, 1987) . This increased entry requires complement andis greatly reduced by mAbs to CD11b (Blackwell et al .,1985 ; Mosser and Edelson, 1985) . Interestingly, severalgroups have reported that leishmania bind directly to macro-phage receptors in the absence of serum or exogenous com-plement, and that this interaction is also inhibited by mAbsto Mac-1 (Blackwell et al ., 1985 ; Mosser and Edelson,1985 ; Russel and Wright, 1988 ; Tàlamus-Rohana et al .,1990 ; Cooper et al ., 1988) . Some of these groups (Russelland Wright, 1988; Talamus-Rohana et al ., 1990) suggest thatleishmania surface molecules may bind directly to Mac-1and perhaps the other leukocyte integrins. A potential criti-cism ofthese experiments is that even in the absence ofexog-enous serum, macrophages may produce enough endoge-nous C3 to facilitate a complement-dependent parasitebinding (Wozencraft et al ., 1986) . Thus, it is still difficult todiscern whether leishmania binding to macrophages throughthe leukocyte integrins requires complement .To address this question, we developed an assay for leish-

mania binding in which the presence ofopsonic complementcould be rigorously controlled . Parasites were preincubatedin the presence or absence ofcomplement and allowed to ad-here to both transfected fibroblasts expressing members ofthe leukocyte integrin family (Mac-1 and LFA-1) or to im-

Figure 8. The effectof purifiedleishmania LPG on the bind-ing ofL. major promastigotesto Mac-1 plates . (A) Increas-ing concentrations of preop-sonized leishmania promasti-gotes were added to Mac-1plateswhich had been pretreat-ed for 15 min with 100 itg/mlpurified LPG (9) or saline(o) . The LPG was left in thewells during the incubationwith leishmania . (B) A total

x 10 7 promastigotes were incubated in 5% C8D serum and increasing concentrations (0-500 ug/ml) of leishmania LPG for 15 min .A constant amount of unwashed promastigotes (2 x 106) was added to untreated Mac-1 plates in triplicate (o) . The binding of an equalnumber of parasites incubated in 20% C8D and 500 Ag/ml LPG is included (*) .

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munoaffinity-purified leukocyte integrin substrates (Mac-1,LFA-1, and p150,95) . Our results suggest that leishmaniapromastigotes require opsonic complement to bind to theleukocyte integrins . Furthermore, we find that opsonizedleishmania bind predominantly to the Mac-1 integrin . Theydo not bind appreciably to LFA-1 and bind only weakly top150,95 .Our evidence for these conclusions is as follows : (a) Two

species of leishmania (L. major and L. amazonensis) boundstrongly to purified Mac-1 substrates but not to those con-taining LFA-1 or control proteins . Binding to purifiedp150,95 was reproducibly weaker, but was partially inhibitedby specific mAb (data not shown) . All three purified recep-tors retain their antigenicity and biological function (Dustinand Springer, 1989 ; Diamond et al ., 1990 ; Stacker andSpringer, 1991), and the site densities used in this study weresufficient to sustain endothelial cell binding (data not shown) .(b) The binding to Mac-1 was dose-dependent both for theconcentration ofparasites and Mac-1 . It was also specific be-cause it was inhibited strongly by some but not all mAbs toMac-1 . (c) L. major showed an enhanced ability to bind toCOS cells expressing Mac-1, but not to LFA-1 or mock-trans-fected cells . This increase was abolished by mAbs to Mac-1 .The low-level background binding of promastigotes to un-transfected and LFA-1-transfected COS cells presumablyoccurred by a serum-independent mechanism which maybe mediated via lectin-like receptors on mammalian cells

Figure 7. The inhibitionofL. majorpromasti-gote binding to Mac-1 substrates . Mac-1 sub-strates were incubated with mAbs for 20 minbefore and during the addition of 2 x 106leishmania in 5% C8D for 60 min at 35°C .Antibodies LPM19C, 904, and 14136E .2were used as ascites diluted 1 :100. AntibodiesR6.5, IB4, LM2/1, and OKMI were used aspurified IgG at a final concentration of 10Ag/ml . R6.5 mAb to ICAM-1 was used as anonbinding control antibody. Counts perminute were determined in triplicate andcompared to simultaneous control counts perminute (no antibody) . Results reported hereare the mean percent control of at least twoexperiments done in triplicate .

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Figure 9 The binding of radiolabeled L. major promastigotes totransfected COS cells . Increasing concentrations ofpromastigoteswere added to COS cells transfected with Mac-1 (0,e), with LFA-1(e,A), or with plasmid alone (o,m) in the presence (closed sym-bols) or absence (open symbols) of 5% C8D. Binding of promasti-gotes in serum to Mac-1 substrates in the presence of antibody toMac-1 (LPM19C) (v) or to LFA1 (TSl/22) (v) is included . Datapoints represent the mean (triplicates) cpm/well f SD (left axis)and the deduced number of parasites per cell .

(Blackwell et al ., 1985 ; Wilson and Pearson, 1988) recog-nizing LPGs on leishmania (Bouvier et al ., 1985 ; Handmanand Goding, 1985) . (d) The binding of leishmania topurified Mac-1 or to COS cells expressing Mac-1 requiredthe presence ofcomplement, since incubation in the absenceof serum, in the presence of heat-inactivated serum, or withserum immunologically or biochemically depleted of C3eliminates binding . The addition of C3 to depleted serumpartially restored binding to purified Mac-1 . Failure to com-pletely restore binding was due to complement-mediated ly-sis ofpromastigotes after C3 addition, as the C3-deficient se-rum used contains all the terminal components of the

complement cascade . Lysed promastigotes release radio-label (Mosser and Edelson, 1984), which was removed bywashing . (e) These experiments with transfected and purifiedMac-1 agree with data from human macrophages, as thepresence of active complement greatly enhances binding ofparasites, and this increase was inhibited almost completelyby blocking mAbs to Mac-1, and not to LFA-1 or p150,95.Interestingly, in our system, the complement receptor type 1(CRI.) apparently has only a minor role in promastigote bind-ing . Monoclonal antibodies to the CR3 (OKMI and LMP-19c) block the majority ofparasite binding to human macro-phages, whereas mAbs to the CRl block poorly, if at all .The requirement for opsonic complement for leishmania

binding to Mac-1 suggests that the interaction between para-site and macrophage may be mediated by Mac-1 binding toiC3b, a well-characterized adhesive interaction that is criti-cal to immune clearance of foreign particles (Beller et al .,1982 ; Wright et al ., 1983) . The mAbs to Mac-1 which in-hibit leishmania binding are the same as those which blockiC3b binding to cellular or purified Mac-1 (Diamond et al .,1989 ; Garcia-Aguilar, J ., M . S . Diamond, andT. A. Springer,manuscript in preparation) : LPM19c and 14B6E.2 blockstrongly, OKM1 inhibits partially, and LM2/1 blocks onlyweakly the binding of leishmania to Mac-1 . Parasites whichbind to intact macrophages without a requirement for exog-enous complement may be able to fix the small amount ofC3 that is generated endogenously by macrophages . In con-trast, others have demonstrated that beads coated with iso-lated leishmania molecules bound to neutrophils in the ab-sence of complement (Talamus-Rohana et al ., 1990) . Thisbinding was inhibited by two mAbs (904 and IB4) that reactwith Mac-1 at sites distinct from the iC3b binding site . Webelieve that this serum-independent binding represents a mi-nor contribution, because when we use intact viable para-sites, these two antibodies have little effect on binding to

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Figure 10. Photomicrograph show-ing the binding ofL. majorpromasti-gotes to COS cells expressing Mac-1 .A total of 2.5 x 106 leishmaniawere added to 24-well plates contain-ing N5 x 10° COS cells in the pres-enceof5 % C8D. Numerous promas-tigotes bound to the periphery ofsome cells are indicated by arrows .

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Mac-1 . The fixation of complement by the two leishmaniasurface structures gp63 and LPG may explain in part whyother groups have observed a role for these structures in thebinding of leishmania to macrophages (Talamus-Rohana etal ., 1990 ; Russell and Wright, 1988) . In our experiments,the inhibition ofleishmaniabinding to Mac-1 by LPGdid notoccur unless LPG was coincubated with parasites and com-plement . This inhibitory effect occurred at relatively highLPG concentrations and was overcome by the addition ofex-cess serum . Thus, we believe that the inhibitory role ofsoluble LPG in our system is due, at least in part, to its abil-ity to fix and consume complement .Using purified proteins and transfected cells, we have ex-

amined the interaction ofleishmania with individual cell sur-face receptors and conclude that there is a requirement foropsonic complement for parasite interaction with Mac-1 . Itis interesting that several other microorganisms have beenobserved to bind to Mac-1 including E. coli (Wright andJong, 1986), B. pertussis (Relman et al ., 1990), and H. cap-sulatum (Bullock and Wright, 1987) . These studies wereperformed using macrophages as the source ofMac-1 . It willbe interesting to determine whether these organisms binddirectly to Mac-1 or, in a manner similar to leishmania, re-quire opsonic complement to allow adhesion . We hypothe-size that many intracellular microbes may subvert the well-characterized Mac-1-iC3b phagocytosis pathway to initiatecellular infections.

The authors would like to thank Dr . Emanuela Handman for providing theLPG and for her critical reading of the manuscript.

This work was supported by National Institutes of Health grantsT32GM07753-11, CA31799, and AI24313 .

Received for publication 25 June 1991 and in revised form 7 October 1991 .

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